Methionine Restriction Impairs Degradation of a Protein that Aberrantly Engages the Endoplasmic Reticulum Translocon

Proteins that persistently engage endoplasmic reticulum (ER) translocons are degraded by multiple translocon quality control (TQC) mechanisms. In Saccharomyces cerevisiae , the model translocon-associated protein Deg1 -Sec62 is subject to ER-associated degradation (ERAD) by the Hrd1 ubiquitin ligase and, to a lesser extent, proteolysis mediated by the Ste24 protease. In a recent screen, we identified nine methionine-biosynthetic genes as candidate TQC regulators. Here, we found methionine restriction impairs Hrd1-independent Deg1 -Sec62 degradation. Beyond revealing methionine as a novel regulator of TQC, our results urge caution when working with laboratory yeast strains with auxotrophic mutations, often presumed not to influence cellular processes under investigation.

(A) Following persistent aberrant translocon engagement, Deg1-Sec62 degradation is promoted by the Hrd1 ubiquitin ligase (in concert with the Ubc7 ubiquitin-conjugating enzyme and its membrane anchor Cue1) and, to a lesser extent, by the zinc metalloprotease Ste24 and at least one additional mechanism (Runnebohm et al. 2020).Deg1-Sec62 undergoes N-linked glycosylation after aberrant translocon engagement (Rubenstein et al. 2012).Ub, ubiquitin.(B) Gene Ontology (GO) Process Terms (and associated genes) enriched in screen for requirements for Deg1-Sec62 degradation are listed.GO Terms were identified with the Gene Ontology Term Finder at the Saccharomyces Genome Database (www.yeastgenome.org/goTermFinder)using a P value cutoff of 0.01.Adapted from (Turk et al. 2023).(C) Cycloheximide chase of yeast of the indicated genotypes expressing Deg1-Sec62 cultured in synthetic defined (SD) medium containing methionine.H, HRD1.M, MET15.mΔ, met15Δ.(D, E, F) Yeast of the indicated genotypes expressing Deg1-Sec62 or Deg1-Vma12 were cultured to exponential phase in SD medium containing methionine, followed by a shift to fresh SD medium containing or lacking methionine, as indicated, for two hours before cycloheximide chase.

Description
Many endomembrane system and secreted proteins cross the eukaryotic endoplasmic reticulum (ER) membrane by passing through protein-conducting translocon channels.Proteins that persistently engage or clog translocons are targeted for degradation by one of several conserved translocon quality control (TQC) mechanisms (reviewed in (Wang and Ye 2020)).Unresolved translocon clogging may cause or exacerbate metabolic and neurological disease (Colin et al. 2016;Muona et al. 2016;Kayatekin et al. 2018).The model engineered Deg1-Sec62 protein aberrantly engages translocons in Saccharomyces cerevisiae when its N-terminal tail aberrantly loops post-translationally into translocons and forms a stabilizing disulfide bond with the translocon interior (Rubenstein et al. 2012).Deg1-Sec62 is targeted for degradation by the ER-associated degradation (ERAD) ubiquitin ligase Hrd1 and, to a lesser extent, the zinc metalloprotease Ste24 and at least one uncharacterized mechanism (Figure 1A) (Rubenstein et al. 2012;Runnebohm et al. 2020).
To identify novel genes required for Deg1-Sec62 degradation, we recently conducted and reported a genome-wide, yeast growth-based reporter screen (Turk et al. 2023).Characterization of a subset of genes identified in this screen demonstrated that ERAD is broadly sensitive to perturbed lipid biosynthesis.Further, we found Deg1-Sec62 degradation is impaired by loss of the microtubule motor protein, Kar3, and, to a modest extent, of the histone methyltransferase Set2.Gene Ontology (GO) analysis (www.yeastgenome.org) of 128 candidate TQC-regulating genes identified in the screen revealed significant enrichment of genes linked to five processes associated with sulfur metabolism (Figure 1B).No additional Function, Component, or Process GO terms were enriched.Nine of the eleven genes in these categories encode enzymes in the methionine biosynthesis pathway.
Here, we tested the hypothesis that methionine is required for efficient Deg1-Sec62 degradation.We conducted cycloheximide chase experiments (Buchanan et al. 2016) to compare degradation of the steady state population of Deg1-Sec62 in yeast with wild type and deletion alleles of HRD1 and MET15 in Synthetic Defined (SD) media containing 0.001% methionine.MET15 encodes O-acetyl homoserine-O-acetyl serine sulfhydrylase, which catalyzes synthesis of homocysteine, an immediate precursor of methionine.MET15 is an auxotrophic marker gene deleted in several commonly used laboratory strains of S. cerevisiae (Brachmann et al. 1998).As previously observed (Engle et al. 2017;Runnebohm et al. 2020;Turk et al. 2023), loss of HRD1 slowed Deg1-Sec62 degradation (Figure 1C).By contrast, MET15 deletion did not impair degradation or exacerbate the degradation defect of yeast lacking HRD1 under these conditions.
We considered the possibility that methionine in the growth medium suppresses an effect of MET15 deletion and assessed Deg1-Sec62 degradation in MET15 and met15Δ yeast in the context of methionine restriction.Yeast cultured to exponentialphase growth in medium containing methionine were shifted to fresh medium containing or lacking methionine for two hours (Figure 1D) prior to cycloheximide chase analysis.The rate of Deg1-Sec62 turnover was not altered regardless of the presence or absence of the MET15 gene or of methionine in the medium.
We hypothesized methionine availability impacts Hrd1-independent TQC.Given the major role of Hrd1 in TQC, an effect of methionine on Hrd1-independent degradation may be masked by the presence and activity of Hrd1.Consistent with this, we observed that, despite reduced steady state abundance, two hours of methionine restriction significantly stabilized Deg1-Sec62 in hrd1Δ met15Δ yeast (but not hrd1Δ MET15 yeast) (Figure 1E).After one hour, 69% of Deg1-Sec62 remained in methionine-restricted hrd1Δ met15Δ yeast, compared to 35% in hrd1Δ met15Δ yeast in media containing methionine.Thus, methionine restriction impairs at least one Hrd1-independent TQC degradative mechanism that acts on Deg1-Sec62.
Several potential mechanisms may explain how methionine restriction impairs TQC degradation of Deg1-Sec62.We have previously shown that STE24 deletion stabilizes and reduces steady state abundance of Deg1-Sec62 to a similar extent as methionine-restricted hrd1Δ met15Δ yeast (Runnebohm et al. 2020).This observation is consistent with methionine restriction and STE24 deletion stabilizing Deg1-Sec62 via a common mechanism.Ste24 cleaves both prenylated CAAX box-containing proteins and non-prenylated proteins (Hildebrandt et al. 2016).It is unknown whether Ste24 directly cleaves Deg1-Sec62, which does not have a CAAX box.Maturation of cleaved, prenylated CAAX box substrates includes methylation of the newly exposed C-terminus, using the methyl donor S-adenosylmethionine, for which methionine is a direct precursor (Cherest and Surdin-Kerjan 1978;Hrycyna et al. 1991).Methionine depletion might disrupt maturation of a prenylated Ste24 substrate that promotes TQC degradation of Deg1-Sec62.Alternatively, methylation of translocon-associated proteins such as Deg1-Sec62 may facilitate direct recognition by Ste24 or other as-yet uncharacterized TQC machinery.Indeed, mass spectrometry analysis demonstrated methylation of two lysine residues within Deg1-Sec62 (see Supplemental Information in (Engle et al. 2017)).Future experiments will assess the requirement of Deg1-Sec62 methylation for its TQC degradation.
The effect of methionine restriction on TQC could be multi-faceted or indirect.The yeast response to methionine restriction is mediated in large part by the Skp-Cullin-F Box (SCF) ubiquitin ligase complex SCF Met30 (reviewed in (Lauinger and Kaiser 11/9/2023 -Open Access 2021)).A shift in SCF Met30 substrate specificity during methionine stress results in transcriptional reprogramming and cell cycle arrest.Our genome-wide screen revealed a modest Deg1-Sec62 degradation defect in yeast lacking SET2, which encodes a histone methyltransferase (Strahl et al. 2002;Turk et al. 2023).Further, loss of phospholipid methyltransferases Cho2 or Opi3 partially stabilizes Deg1-Sec62 (Henry et al. 2012;Turk et al. 2023).Thus, depletion of methionine and, by extension of S-adenosylmethionine, may impair degradation by altering SCF Met30 substrate specificity, gene expression patterns, or membrane lipid composition.In any case, the impacts of dampened methylation on degradation would need to occur on the relatively short timescale of two hours (the period of methionine restriction in our experiments).
Despite impaired degradation, steady state abundance of Deg1-Sec62 was reduced in hrd1Δ met15Δ yeast following methionine restriction (Figure 1E), consistent with a diminished rate of synthesis.A similar reduction in Deg1-Vma12 abundance was not observed under these conditions (Figure 1F), even though expression of genes encoding both protein quality control substrates were driven by the GPD promoter.Further investigation will be required to understand how methionine restriction preferentially impacts steady state abundance of this TQC substrate.Unaltered Deg1-Vma12 degradation kinetics in methionine-restricted hrd1Δ met15Δ yeast indicates that altered Deg1-Sec62 abundance and degradation are not caused by broadly impaired ubiquitin-proteasome system or reduction in cell viability.
Combined with other recent studies (Oss et al. 2022), our results suggest caution is warranted when working with auxotrophic laboratory yeast strains, including those with MET15 mutations.Auxotrophic mutations are often presumed to have negligible influence on cellular processes under investigation.Our experiments provide a counterexample.In the present case, the impact of impaired methionine synthesis by MET15 deletion was mitigated by the presence of methionine in yeast growth medium.Given that many microbial experiments are conducted in growth medium containing low concentrations of nutrients whose synthesis may be impaired by auxotrophic gene mutation, this may not always be the case.Several genes encoding enzymes in the methionine biosynthesis pathway were identified in our yeast growth-based screen for requirements of Deg1-Sec62 degradation (Turk et al. 2023), suggesting that methionine may have become sufficiently depleted during the screen to allow for observable changes in growth in these mutants.Future experiments may directly test the requirement of other methionine and S-adenosylmethionine biosynthetic genes for TQC.
A failure to unclog stalled translocons reduces cellular health and may contribute to human disease (Ast et al. 2016;Colin et al. 2016;Muona et al. 2016;Kayatekin et al. 2018;Runnebohm et al. 2020).Cells have developed multiple partially overlapping mechanisms to resolve clogged translocons, reflecting the critical nature of protein movement across biological membranes.We reported here that methionine restriction impedes Hrd1-independent TQC.Methionine is an essential amino acid in humans.Deficiency or imbalance of methionine contributes to several chronic liver diseases (reviewed in (Li et al. 2020)).Our results suggest patients suffering from such conditions may experience disrupted hepatic TQC.A better understanding of the relationship between methionine physiology and degradation of translocon-clogging proteins could lead to improved therapeutic strategies for these maladies.
Cycloheximide Chase, Cell Lysis, and Western Blotting.Cycloheximide chase experiments were performed using exponential-phase cultures as described (Buchanan et al. 2016).For methionine restriction experiments, yeast were cultured to exponential growth in medium containing methionine.Two x 10 OD 600 units (1 OD 600 unit is 1 mL of yeast at an OD 600 of 1.0) were harvested and centrifuged at 7000 x g for 5 min at room temperature.One cell pellet from each pair was washed three times with and resuspended in fresh medium with methionine.The other cell pellet was washed three times with and resuspended in fresh medium lacking methionine.Cultures were incubated for 2 hours at 30°C prior to cycloheximide chase.The presence or absence of methionine was maintained throughout the cycloheximide chase experiment.Protein extracts were prepared and analyzed by western blotting as described (Kushnirov 2000;Watts et al. 2015).At their C-termini, Deg1-Sec62 and Deg1-Vma12 possess two copies of the Staphylococcus aureus Protein A tag, which binds to mammalian immunoglobulins (Hjelm et al. 1972); thus, Deg1-Sec62 and Deg1-Vma12 were directly detected with AlexaFluor-680-conjugated rabbit-anti-mouse antibodies (Life Technologies, Inc.; 1:40,000).Pgk1 was detected with mouse antiphosphoglycerate kinase 1 antibodies (clone 22C5D8; Life Technologies, Inc.; 1:20,000), followed by AlexaFluor-680conjugated rabbit-anti-mouse antibodies (1:40,000).An Odyssey CLx Infrared Imaging System and Empiria Studio Software (Li-Cor) were used to image and analyze membranes.The (Deg1-Sec62 or Deg1-Vma12)/Pgk1 signal intensity ratio was used to normalize protein abundance in a given experiment.Percent remaining was determined by comparing the (Deg1-Sec62 or Deg1-Vma12)/Pgk1 signal intensity ratio at each time point to this ratio at 0 minutes for each culture.
Statistical Analysis.Data were analyzed using GraphPad Prism (version 10).Statistical analyses are described in the figure legend.

Reagents
Yeast strains used in this study.This study

Figure 1 .
Figure 1.Methionine restriction impairs Hrd1-independent degradation of Deg1-Sec62: For (C) and (D), means of percent Deg1-Sec62 remaining for two biological replicates are plotted.Error bars indicating range are included, except for when the error bar would be shorter than the symbol.For (E), means of percent Deg1-Sec62 remaining for three (hrd1Δ MET15) or six (hrd1Δ met15Δ) biological replicates are plotted.Error bars indicate standard error of the mean.Means of percent Deg1-Sec62 remaining at 60 minutes in (E) were evaluated by one-way ANOVA (F, 9.290; P, 0.0012) followed by Tukey's multiple comparison test; adjusted pairwise P values are included beneath the plot.Pgk1, loading control.For (F), values for one experiment are plotted.